1. Field of the Invention
The present application relates generally to the use of hybridization chain reaction to create RNA polymers that are able to activate the RNA-dependent kinase PKR.
2. Description of the Related Art
Hybridization Chain Reaction (HCR) is a method for the triggered hybridization of nucleic acid molecules, typically starting from metastable, monomer hairpins. HCR is described, for example, in U.S. patent application Ser. No. 11/087,937, filed Mar. 22, 2005 (published as U.S. Publication No. 2005-0260635 on Nov. 24, 2005), and Dirks and Pierce (Dirks, R. and N. Pierce, Proc. Natl. Acad. Sci. USA 101(43): 15275-15278 (2004)), which are incorporated herein by reference in their entirety. In a simple version of this process, metastable monomer hairpins undergo a chain reaction of hybridization events to form a nicked helix when triggered by a nucleic acid initiator strand. A fundamental principle behind HCR is that short loops are resistant to invasion by complementary single-stranded nucleic acids. This stability allows for the storage of potential energy in the form of loops; potential energy is released when a triggered conformational change allows the single-stranded bases in the loops to hybridize with a complementary strand. One embodiment of HCR is illustrated in FIG. 1.
HCR can be used for a wide variety of purposes, for example, to create a visual signal that identifies the presence of one or more target analytes in a sample using nano-gold particles, as described by U.S. patent application Ser. No. 11/371,347, filed on Mar. 7, 2006, herein incorporated by reference. It can also be used for in situ imaging and detection applications, as described by U.S. patent application Ser. No. 11/371,346, filed on Mar. 7, 2006, herein incorporated by reference. As described in detail below, it has now been found that HCR can be used to create RNA polymers in target cells, thereby activating the protein kinase PKR.
Protein kinases and other cell-signaling messengers are activated during an immune response. In vitro studies show that PKR is activated when two PKR molecules dimerize and phosphorylate each other in complex with RNA duplexes longer than approximately 30 bp, and that the strength of the activation increases with duplex length up to ˜85 bp (Manche, L. et al. Molecular and Cellular Biology 12:5238-5248 (1992); Wu, S., and R. J. Kaufman. The Journal of Biological Chemistry 272:1291-1296 (1997); which are incorporated herein by reference in their entirety). Activation of PKR can lead to selective cell death, which is a strategy envisioned for the development of therapies for cancer (Shir, A. and A. Levitski. Nature Biotechnology 20:895-900 (2002); Friedrich, I. et al. Serminars in Cancer Biology 14:223-230 (2004); Friedrich, I. et al. Molecular Therapy 12:969-975 (2005); which are incorporated herein by reference in their entirety). Exploiting these activation properties, it was demonstrated that binding of anti-sense RNA to a region that spans the splice point of an oncogenic mRNA fusion entity resulted in creation of a 39-base pair duplex within cancer cells, selectively killing them (Shir, A., and A. Levitzski. 2002). The 39-bp duplex formed by the oncogenic fusion entity and the anti-sense RNA partially activated the protein kinase PKR, which is involved in inhibition of protein synthesis and cell death. In healthy cells, binding between the anti-sense RNA and the non-fused wild-type mRNA yielded only a 20-bp or 19-bp duplex, which resulted in minimal PKR activation. In this approach, the duplex formed in the process of oncogene detection served as the binding site for the activation of PKR. As a result, in this approach, the length of the activating RNA duplex is limited to twice the length of the longest duplex that does not activate PKR; this in turn limits the extent of PKR activation and the efficacy of a corresponding therapy.
A new approach to selective PKR activation has been developed based on the mechanism of HCR. Metastable RNA hairpins interact upon exposure to a target molecule to form long nicked polymers that subsequently activate PKR. The target molecule is preferably a mutant mRNA molecule associated with cancer. Activation of PKR leads to the inhibition of protein synthesis and to cell death (Jagus, R. et al. The International Journal of Biochemistry 31:123-138 (1999); Williams, B. R. G. Oncogene 18:6112-6120 (1999); which are incorporated herein by reference in their entirety). By transducing a detection binding event into the formation of a distinct activation domain, HCR hairpins can be used that bind minimal nucleic acid sequences but generate longer activation duplexes with multiple PKR binding domains (FIG. 5), leading to greater specificity and increased PKR activity. As a result, PKR is more effectively activated in target cells since the length of the activation duplexes is not limited as it is in the prior art approach described above. HCR is thus envisioned as a superior approach for activation of PKR for the purpose of triggering the death of diseased cells.